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http://dx.doi.org/10.2147/DDDT.S72127
Koenimbin, a natural dietary compound of Murraya koenigii (l) spreng: inhibition of McF7 breast cancer cells and targeting of derived McF7 breast cancer stem cells (cD44+/cD24-/low): an in vitro study
Fatemeh ahmadipour1
Mohamed ibrahim noordin1
syam Mohan2
aditya arya1
Mohammadjavad Paydar3
chung Yeng looi3
Yeap swee Keong4
ebrahimi nigjeh siyamak4
somayeh Fani1
Maryam Firoozi5
chung lip Yong1
Mohamed aspollah sukari6
Behnam Kamalidehghan1
1Department of Pharmacy, Faculty of Medicine, University of Malaya, Kuala lumpur, Malaysia; 2Medical research center, Jazan University, Jazan, Kingdom of saudi arabia; 3Department of Pharmacology, Faculty of Medicine, University of Malaya, Kuala lumpur, Malaysia; 4UPM-MaKna cancer research laboratory, institute of Bioscience, Universiti Putra Malaysia, serdang, Malaysia; 5Department of Medical genetics, national institute for genetic engineering and Biotechnology, Tehran, iran; 6Department of chemistry, Faculty of science, Universiti Putra Malaysia, serdang, Malaysia
Background: Inhibition of breast cancer stem cells has been shown to be an effective therapeutic
strategy for cancer prevention. The aims of this work were to evaluate the efficacy of koenimbin,
isolated from Murraya koenigii (L) Spreng, in the inhibition of MCF7 breast cancer cells and
to target MCF7 breast cancer stem cells through apoptosis in vitro.
Methods: Koenimbin-induced cell viability was evaluated using the MTT (3-(4,5-dimethylthiazol-
IntroductionMurraya koenigii (L) Spreng (known as Surabhinimba in Sanskrit), known locally as the
curry leaf, is a member of the Rutaceae family and is widely distributed in South Asia.1
correspondence: Behnam KamalidehghanDepartment of Pharmacy, Faculty of Medicine, University of Malaya, Jalan Universiti, 50603 Kuala lumpur, MalaysiaTel +60 37967 7897Fax +60 37967 4964email [email protected]
Journal name: Drug Design, Development and TherapyArticle Designation: Original ResearchYear: 2015Volume: 9Running head verso: Ahmadipour et alRunning head recto: Koenimbin and inhibition of breast cancer cellsDOI: http://dx.doi.org/10.2147/DDDT.S72127
Note: results are shown as the mean ± standard deviation of three independent experiments.
Figure 1 MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. growth curve for koenimbin-treated McF7 cells at 24, 48, and 72 hours.
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Figure 2 MCF7 cancer stem cells were identified by expression of CD44+ and low expression of cD24-/low in quadrant analysis (cD44+/cD24-/low). each experiment was performed three times (n=3). Abbreviations: FITC, fluorescein isothiocyanate; PE, phycoerythrin.
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Figure 3 Mammosphere formation and Aldefluor™ assay of MCF7 cancer stem cells. Notes: size of mammospheres containing McF7 cancer stem cells on day 5 (A) and day 7 (B). McF7 cancer stem cells were cultured in mammosphere-forming conditions, and were incubated with koenimbin (0, 1, 2, and 4 µg/mL) for 7 days (magnification ×100) (C). Koenimbin reduced the size of the primary mammospheres. in the absence of drug, the second and third passages derived from koenimbin-treated primary mammospheres yielded smaller numbers of spheres in comparison with the control. The size of the mammospheres was estimated using V = (4/3)πr3. Koenimbin inhibits mammosphere formation and prevents self-renewal of (D) primary, (E) secondary, and (F) tertiary mammosphere-forming units. Data are shown as the mean ± standard deviation (n=3). **P0.01 versus control. (G) Aldefluor assay of MCF7 cancer stem cells. single cells obtained from cell cultures were incubated for 50 minutes at 37°C in Aldefluor assay buffer containing an ADH substrate, BODIPY-aminoacetaldehyde (1 µmol/l per 1×106 cells). a cell population (r2) with high aDh activity was reported to enrich mammary stem/progenitor cells. (H) inhibitory effect of koenimbin on aDh-positive cell populations. McF7 cancer stem cells were treated with koenimbin 1, 2, or 4 µg/mL for 4 days and subjected to Aldefluor assay and flow cytometry analysis. Koenimbin decreased the percentage of aDh-positive cells. Data are shown as the mean ± standard deviation (n=3). *P0.05 versus control; **P0.01 versus control. Abbreviations: aDh, aldehyde dehydrogenase; K, koenimbin.
Notes: cells were exposed to koenimbin at various concentrations (0, 2.5, 5, or 10 µg/ml) and incubated for 24 hours. The table summarizes the percentages of cells in each phase of the cell cycle after treatment with koenimbin. Data in the same vertical column but different rows refer to the same phase of the cell cycle and different koenimbin concentrations. *Indicates a significant difference (P0.05). Data are shown as the mean ± standard deviation (n=3).
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Figure 4 cell cycle histograms from analyses of McF7 cells treated with 0 (A), 2.5 (B), 5 (C), and 10 µg/ml (D) of koenimbin for 12 hours. (E) summary of cell cycle progression for control and koenimbin-treated McF7 cells.Notes: Data are shown as the mean ± standard deviation (n=3). *P0.05 versus control.
alteration of all cell cycle phases (Table 2). A dramatic
increase in the sub-G0 phase, which indicates DNA frag-
mentation, and reduction of the G0/G1, S, and G2/M phases
was observed in cells treated with koenimbin 10 µg/mL.
However, no significant difference was observed in cells
treated with koenimbin 2.5 or 5 µg/mL (Figure 4A–E).
Therefore, the cell cycle analysis indicated significant cyto-
toxicity and cell inhibitory effects of koenimbin in MCF7
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effects of koenimbin on membrane permeability, MMP, and cytochrome c releaseAs the main source of cellular reactive oxygen species and
adenosine triphosphate, the mitochondria play an impor-
tant regulatory role in controlling the survival and death
of cells. We used MMP fluorescent probes to examine the
function of mitochondria in treated and untreated MCF7
cells. As shown in Figure 5, the untreated cells were
strongly stained with MMP dye in comparison with cells
treated with koenimbin 9 µg/mL for 24 hours. The reduc-
tion in MMP fluorescence intensity indicated that MMP
was destroyed in the treated cells. A significant increase
in cell membrane permeability was also observed in the
treated cells after 24 hours of treatment with koenimbin.
Twenty-four hours of exposure to koenimbin also resulted
in an increase in cytochrome c in the cytosol when com-
pared with the control.
Bioluminescent assays for caspase activityExcessive production of reactive oxygen species from the
mitochondria and collapse of MMP may activate down-
stream caspase molecules, leading to apoptotic cell death.
To examine this, we measured the bioluminescent intensi-
ties relating to caspase activity in the MCF7 cells treated
with different concentrations of koenimbin for 24 hours. As
shown in Figure 6, a significant dose-dependent increase in
caspase-7 and caspase-9 activity was detected in the treated
cells, while no marked change in caspase-8 activity was
observed between treated and untreated cells. Hence, apop-
tosis induced by koenimbin in MCF7 cells is mediated via
the intrinsic mitochondrial caspase-9 pathway and not the
extrinsic death receptor-linked caspase-8 pathway.
Translocation of nF-κBNF-κB is a transcription factor critical for cytokine gene
expression. Activation of NF-κB in response to inflammatory
Hoechst
Control
Koenimbin(9 µg/mL)
Membranepermeability MMP Cytochrome c Merged
Figure 5 representative images of McF7 cells treated with medium alone and koenimbin 9 µg/ml, and stained with hoechst for nuclear, cytochrome c, membrane permeability, and MMP dyes, and cytochrome c dye. The images from each row are obtained from the same field of the same treatment sample (magnification 20×). Abbreviation: MMP, mitochondrial membrane potential.
4
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Concentration of K (µg/mL)3.7
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**
**
*
*
Figure 6 relative bioluminescence expression of caspase-7, caspase-8, and caspase-9 in McF7 cells treated with koenimbin at various concentrations.Notes: The results are shown as the mean ± standard deviation of three independent experiments. Statistical significance is expressed as *P0.05. Abbreviation: K, koenimbin.
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Koenimbin and inhibition of breast cancer cells
cytokines, such as TNF-α, mediates nuclear migration to
enable DNA-binding activity and facilitate target gene expres-
sion. As seen in Figure 7A, koenimbin suppressed the trans-
location of cytoplasmic NF-κB to the nucleus. The significant
decline in nuclear NF-κB translocation in TNF-α-stimulated
MCF7 cells treated with koenimbin as shown by the statisti-
cal analysis confirms the inhibitory activity of the compound
against nuclear translocation of NF-κB (Figure 7A and B).
A Nucleus
Untreated
Untreated +1 ng/mL TNF-α
Curcumin 50 µM +
1 ng/mL TNF-α
NF-κB Merged
Koenimbin15 µg/mL +
1 ng/mL TNF-α
B400
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leus
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esce
nt in
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Figure 7 (A) Photographs of intracellular targets in stained McF7 cells treated with koenimbin for 3 hours and then stimulated for 30 minutes with TnF-α 1 ng/ml (nF-κB activation). (B) Representative bar chart indicating a significant decline in average fluorescent intensity of nuclei NF-κB, confirming that koenimbin inhibited TNF-α-induced translocation of nF-κB from the cytoplasm to the nucleus.Notes: Data are shown as the mean ± standard deviation (n=3). *P0.05 versus control. Abbreviations: nF-κB, nuclear factor kappa B; TnF-α, tumor necrosis factor alpha.
Figure 8 Quantitative analysis of the human apoptosis proteome profiler array in koenimbin-induced MCF7 cells. MCF7 cells were lysed and protein arrays were performed. cells were treated with koenimbin 9 µg/ml for 24 hours and total cell protein was extracted. equal amounts (300 µg) of protein from each control and treated sample were used for the assay. Quantitative analysis of the arrays showed differences in the apoptotic markers. Notes: The graph shows the difference between treated cells as well as untreated control cells (A). representative images of the apoptotic protein array are shown for the control (B), treated (C), and the exact protein name of each dot in the array (D). The results are shown as the mean ± standard deviation for three independent experiments. *Indicates a significant difference from control (P0.05).
effect of koenimbin on apoptotic markersAfter exposure of MCF7 cells to koenimbin for 24 hours, the
cells were lysed and apoptotic markers were examined using
a human apoptosis protein array. In Figure 8A–D, the images
represent the changes of apoptotic markers in treated and
untreated cells. The most important markers involved in the
apoptosis signaling pathway, such as Bax, Bcl2, caspase-7,
and caspase-8, were induced, along with cytochrome c.
HSP70, a significant chaperone involved in apoptosis, was
also downregulated in this in vitro model.
Protein expression of apoptotic markers and the Wnt/β-catenin self-renewal pathwayAlthough many proteins associated with apoptosis were
observed to be upregulated or downregulated in the protein
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Koenimbin and inhibition of breast cancer cells
array, proteins such as Bax and HSP70 were significantly
induced.
The role of the mitochondria in modulation of apoptotic
markers at the protein level was examined, and expression
of Bax, a proapoptotic protein, and Bcl2, an antiapoptotic
protein, were increased and decreased, respectively, in
koenimbin-induced MCF7 cells. Further, protein expres-
sion of HSP70 was downregulated in a dose-dependent
manner (Figure 9). Additionally, treatment of MCF7 cells
with koenimbin decreased expression levels of β-catenin and
cyclin D1 (Figure 10A), while the expression level of p-β-
catenin (Ser33/Ser37/Thr41) increased (Figure 10B). In this
study, MG132 was used to suppress proteasome functional
activity and determine the status of p-β-catenin (Ser33/Ser37/
Thr41) in response to koenimbin. Here, our result indicated
reduced expression of p-GSK3β (Ser9) with increasing
concentrations of koenimbin (Figure 10C). The koenimbin-
induced β-catenin phosphorylation is reversed in the pres-
ence of LiCl, a GSK3β inhibitor (Figure 10D). As shown in
Figure 10D, koenimbin has the ability to decrease LiCl-induced
GSK3β phosphorylation and accumulation of β-catenin.
DiscussionApoptosis is associated with many biochemical changes in
cells, including nuclear fragmentation, change in the MMP,
and regulation of caspases.48 The present study is the first
report on the in vitro effects of koenimbin, a natural com-
pound derived from the plant M. koenigii (L) Spreng, against
MCF7 cells and derived MCF7 stem cells/progenitors. Inter-
estingly, koenimbin inhibited the growth of MCF7 cells and
derived MCF7 stem cells/progenitors, while non-invasive
MCF-10A cells were more resistant to koenimbin-mediated
Figure 9 Western blot analysis of koenimbin in selected apoptotic signaling markers.Notes: The blot densities are expressed as fold of control (A). The HSP70 protein level was also downregulated, showing significant changes on treatment with koenimbin 4 and 8 µg/ml (B). The Bax (C) and Bcl2 (D) apoptotic markers were significantly elevated and reduced respectively, in a dose-dependent manner. The data are shown as the mean ± standard deviation (n=3). *P0.05 versus control. Abbreviation: hsP, heat shock protein.
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A
β-catenin
Cyclin D1
B 4 µg/mL koenimbin (4 days) – + – +
10 µmol/L MG132 – +50 mM LiCl – + – –
p-β-catenin (Ser33/37/Thr41)
C p-GSK3β
GSK3β
D 50 mM LiCl – +
+
– +
+ 4 µg/mL koenimbin (4 days) – –
β-catenin
p-GSK3β
GSK3β
β-actin
+ +
0 1 2
0 1 2 4 µg/mL koenimbin (4 days)
4 µg/mL koenimbin (4 days)
Figure 10 Western blot analysis of the Wnt/β-catenin self-renewal pathway in McF7 cells treated with koenimbin. Koenimbin downregulated this pathway. Notes: Koenimbin decreased protein expression levels of β-catenin and cyclin D1 in McF7 cells (A). Koenimbin increased the phospho-β-catenin ser33/ser37/Thr41, whereas licl suppressed phosphorylation through inactivation of gsK3β (B). Koenimbin decreased the expression level of p-gsK3β, while the protein expression of total gsK3β was unchanged (C). Koenimbin decreased β-catenin and licl-induced gsK3β phosphorylation, while licl elevated the protein expression level of β-catenin through gsK3β phosphorylation (D). each experiment was performed three times (n=3). Abbreviation: gsK, glycogen synthase kinase.
antiproliferative activity than the MCF7 cells and derived
MCF7 CSCs. However, several chemotherapeutic drugs,
including vincristine, vinblastine, and paclitaxel are derived
from plants49 and affect normal cells.50
The cell morphology, cell membrane permeability,
and nuclei area were significantly diminished by treatment
with koenimbin. Due to their role in direct activation of
the apoptotic program in cells, the mitochondria have been
described as key players in the apoptotic process.51 Therefore,
the complex role of the mitochondria in apoptosis of MCF7
cells was investigated by detection of changes in MMP, as it
is assumed that its disruption is the onset of formation of the
mitochondrial membrane transition pore.52 The high content
analysis conducted in this research indicated that koenimbin
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the Wnt/β-catenin self-renewal pathway and cyclin D1,
in which the koenimbin-induced β-catenin at Ser33/37/Thr41
and degradation of the proteasome is possibly via GSK3β
activation in breast cancer cells, as one of the possible
mechanisms to target the MCF7 CSCs.
ConclusionIn conclusion, koenimbin is able to trigger apoptosis in breast
cancer cells in vitro. Treatment of human MCF7 breast cancer
cells with koenimbin resulted in apoptosis, with cell death-
transducing signals regulating MMP by downregulating Bcl2
and upregulating Bax, thereby triggering release of mitochon-
drial cytochrome c to the cytosol. Upon entering the cytosol,
cytochrome c activates caspase-9, then triggers activation of
caspase-7, and consequently cleaves specific substrates lead-
ing to apoptosis through the intrinsic pathway. Additionally,
koenimbin is able to target MCF7 CSCs as determined by
the mammosphere formation assay and Aldefluor assay. Our
study also identified downregulation of the Wnt/β-catenin
self-renewal pathway as one of the possible mechanisms of
action of koenimbin. In conclusion, this study demonstrates
the therapeutic potential of koenimbin in the chemopreven-
tion of breast cancer, and provides a strong rationale for
clinical evaluation of this compound in the future.
AcknowledgmentThe authors would like to express their utmost gratitude
and appreciation to University of Malaya Research Grant
(RG084-13BIO), IPPP grants (PG082-2013B and PG116-
2014A), and BKP grant (BK020-2012) for providing finan-
cial support to conduct this study.
DisclosureThe authors report no conflicts of interest in this work.
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